AMP-dependent protein kinase (AMPK) is activated by energy deprivation and is a critical regulator of cellular energy balance. Activation of AMPK allows cells to survive under conditions of energy stress by turning on ATP-producing catabolic pathways, and inhibiting ATP-consuming anabolic processes. Conversely, the mammalian target of rapamycin complex 1 (mTORC1) is activated by growth factors and nutrients, and promotes anabolic processes leading to cell growth. Recently, activation of AMPK has been implicated in the beneficial, glucose lowering effects of the anti-diabetic drug metformin, which induces energy stress. Thus, elucidation of the downstream functions of metformin and AMPK responsible for these beneficial effects will greatly impact our understanding of, and ability to better control cellular metabolism. To this end, cell culture experiments have indicated that AMPK can inhibit mTORC1 through two independent mechanisms: activation of the Tsc1-Tsc2 complex (an upstream inhibitor of mTORC1), and inhibition of Raptor (a critical component of mTORC1), suggesting a potential role for mTORC1 in this process. Therefore, the main goal of this proposal is to elucidate the key mechanisms that regulate mTORC1 inhibition by metformin and energy stress in the liver, and to determine the metabolic consequences of this regulation. Conditional deletion of Tsc1 in the mouse liver or cultured hepatocytes will provide a genetic model to study the relative contribution of Tsc1-2 to the inhibition of mTORC1 by Metformin, and other forms of energy and nutrient stress. The requirement for mTORC1 inhibition, through Tsc1, for the cellular and physiological effects of metformin will also be addressed using this mouse model. Functional readouts of mTORC1 and AMPK activation will be used to discern their activities under these conditions. Furthermore, the contribution of AMPK to the inhibition of mTORC1 by metformin will be determined using an additional mouse model with conditional deletion of AMPK in hepatocytes. These experiments address important questions that are crucial to our understanding of the cellular and organismal response to energy fluctuations, and are particularly relevant for further elucidation of the mode of action of metformin, the most commonly prescribed anti-diabetic drug. PUBLIC HEALTH RELEVANCE: The results of this study will impact our understanding of the energy-sensing signaling pathways controlling metabolic homeostasis and will provide novel insights into the therapeutic actions of metformin. Such knowledge is critical to our understanding of type-2 diabetes and to the development of new targeted therapeutics.